RNA-exit-channel: target and method for inhibition of bacterial RNA polymerase

Background art

Bacterial infections remain among the most common and deadly causes of human disease. Infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide (Binder et al., Science 284:1311-1313 (1999)). Multi-drug-resistant bacteria now cause infections that pose a grave and growing threat to public health. It has been shown that bacterial pathogens can acquire resistance to first-line and even second-line antibiotics. (See, Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998); Walsh, C.

Nature 406, 775-781; Schluger, N.

Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al.,

Ann. NY Acad. Sci. 953, 88-97). New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.

The present invention provides one such approach, which involves the transcription machinery of bacteria. RNA is synthesized in cellular organisms by a complex molecular machine, known as RNA polymerase ("RNAP"). In its simplest bacterial form, RNAP comprises at least four subunits with a total molecular mass of around 400 kDa. RNAP mediates the transcription of DNA to produce RNA. Bacterial RNAP is a multimeric protein consisting of subunits .alpha..sub.2, .beta., .beta.', and .omega.. An .sigma. factor is required for initiation of transcription by forming a holoenzyme complex.

Transcription involves the following steps (Record et al. 1996): (i) RNAP binds to promoter DNA, to yield an RNAP-promoter closed complex; (ii) RNAP melts .about.14 bp of promoter DNA surrounding the transcription start site, to yield an RNAP-promoter open complex; (iii) RNAP begins synthesis of RNA, typically carrying out multiple rounds of abortive initiation (synthesis and release of RNA products <9-11 nt in length), as an RNAP-promoter initial transcribing complex; and (iv), upon synthesis of an RNA product of a critical threshold length of 9-11 nt, RNAP breaks its interactions with promoter DNA and begins to translocate along DNA, processively synthesizing RNA as an RNAP-DNA elongation complex.

Currently, there are a few known antibiotics that target RNAP, most notably, rifampicin and rifampicin analogs (See Mitchison, D.

Int. J. Tuberculosis Lung Disease 4, 796-806). Rifampicin is the only anti-tuberculosis compound able to rapidly clear infection and prevent relapse. Without rifampicin, treatment lengths must increase from 6 months to at least 18 months to ensure prevention of relapse. Rifampicin acts by specifically inhibiting RNAP (Campbell et al.,

Cell 104, 901-912). Rifampicin binds to a site adjacent to the active center of bacterial RNAP, the exit channel, and physically prevents synthesis of products longer than .about.4 nucleotides. Unfortunately, tuberculosis strains resistant to rifampicin (and rifampicin analogs) are becoming widespread, effectively removing rifampicin from the therapeutic arsenal. There is a need for novel antibiotics that target the same bacterial enzyme as rifampicin, namely RNAP (and thus that have the same biochemical and therapeutic effects as rifampicin). There is also a need to develop methods for identifying antibiotics that interfere with bacterial RNAP.

Recently crystallographic structures have been determined for bacterial RNAP and eukaryotic RNAP II, and, based on the crystallographic structures, biophysical results, and biochemical results, structural models have been proposed for transcription initiation and elongation complexes (Zhang et al.,

Cell 98, 811-824; Cramer et al., (2000), Science 288, 640-649; Naryshkin et al.,

Cell 101, 601-611; Kim et al.,

Science 288, 1418-1421; Korzheva et al.,

Science 289, 619-625; Ebright, R.

J. Mol. Biol. 304, 687-689; Cramer et al.,

Science 292, 1863-1876; Gnatt et al.,

Science 292, 1876-1882; Mekler et al.,

Cell 108, 599-614; Murakami et al.,

Science 296, 1280-1284; Murakami et al.,

Science 296, 1285-1290; Vassylyev et al.,

Nature 417, 712-719; Bushnell et al.,

Science 303, 983-988; Westover et al.,

Science 303, 1014-1016). The structural models include an approximately 30 .ANG. long, 15 .ANG. wide channel, known as the "RNA-exit-channel," that connects the RNAP active-center cleft to the RNAP exterior. In transcription initiation complexes, transcription initiation factors occupy this channel: i.e., initiation factor .sigma. region 3.2 (also known as the ".sigma.R3/.sigma.R4 linker" or ".sigma.3/.sigma.4 linker") in the case of bacterial transcription initiation complexes and transcription initiation factor IIB N-terminal domain in the case of eukaryotic RNAP II transcription initiation complexes. In transcription elongation complexes, the nascent RNA product occupies this channel.

Summary of the invention

Applicant has discovered that a region within the RNAP RNA-exit-channel comprising two short peptide segments of the RNAP .beta. subunit and one short peptide segment of the RNAP .beta.' subunit is conserved in amino-acid sequence in bacterial species, including both Gram-positive bacteria and Gram-negative bacteria. Throughout the following specification, this region is referred to as the "target," and the three short peptide segments collectively are referred to as the "homologous RNA-exit-channel amino-acid sequence." Applicant further has discovered that this region is not conserved, and in fact is radically different, in amino-acid sequence in eukaryotic RNAP, such as human RNAP I, human RNAP II, and human RNAP III. Applicant further has discovered that this region form an approximately 10 .ANG. wide shallow pocket within the wall of the RNAP RNA exit channel.

Accordingly, a first aspect of the present invention is directed to a method for identifying agents that bind to a bacterial RNAP homologous RNA-exit-channel amino-acid sequence, comprising preparing a reaction solution comprising the agent to be tested and an entity containing a homologous RNA-exit-channel amino-acid sequence; and detecting presence or amount of binding. In a preferred embodiment, detection or quantitation of binding is conducted relative to binding of the agent to an entity containing an altered homologous RNA-exit-channel amino-acid sequence.

Another aspect of the present invention is directed to a method for identifying agents that inhibit an activity of bacterial RNAP via binding to a homologous RNA-exit-channel amino-acid sequence. This aspect entails preparing a reaction solution comprising the agent to be tested, a catalytic entity containing a homologous RNA-exit-channel amino-acid sequence, and a substrate for the entity; and determining extent of inhibition of RNAP activity via binding of the agent to the homologous RNA-exit-channel amino-acid sequence.

In some preferred embodiments, binding or inhibition is compared to binding or inhibition by lipiarmycin (Lpm). Lpm is a macrocyclic antibiotic effective against both Gram-positive bacteria and Gram-negative bacteria (Coronelli et al.,

J. Antibiot. 28, 253-259). Lpm functions by inhibiting bacterial RNAP (Sergio et al.,

J. Antibiot. 1975, 543-549; Talpaert et al.,

Biochem. Biophys. Res. Commun. 63, 328-334; Sonenshein et al.,

J. Bacteriol. 132, 73-79; Sonenshein et al.,

J. Mol. Biol. 127, 55-72). The present invention provides that Lpm inhibits bacterial RNAP by binding to a determinant that includes residues within the bacterial RNAP homologous RNA-exit-channel amino-acid sequence. The present invention also provides for the identification of potential antibacterial agents or antibiotics that, because they interact with residues that are conserved in bacterial RNAP, have broad-spectrum antibacterial activity. It also provides for the identification of potential anti-bacterial agents or antibiotics that, because they interact with residues that are not conserved in eukaryotic RNAP, are relatively non-disruptive to normal cellular functions of eukaryotes.

It is anticipated that compounds identified according to the target and method of this invention would have applications not only in antibacterial therapy, but also in: (a) identification of bacterial RNAP (diagnostics, environmental-monitoring, and sensors applications), (b) labeling of bacterial RNAP (diagnostics, environmental-monitoring, imaging, and sensors applications), (c) immobilization of bacterial RNAP (diagnostics, environmental-monitoring, and sensors applications), (d) purification of bacterial RNAP (biotechnology applications), (e) regulation of bacterial gene expression (biotechnology applications), and (f) antisepsis (antiseptics, disinfectants, and advanced-materials applications).

These and other aspects of the present invention will be better appreciated by reference to the following drawings and detailed description.

The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

Brief description of the drawings

FIG. 1A illustrates sequence alignments for the bacterial RNAP homologous RNA-exit-channel amino-acid sequences within rpoB (.beta.) from Escherichia coli; and corresponding residues of Haemophilus influenzae, Vibrio cholerae, Pseudomonas aeruginosa, Treponema pallidum, Borrelia burgdorferi, Xyella fastidiosa, Camploacter jejuni, Neisseria meningitides, Rickettsia prowazekii, Chlamydia trachomatis, Mycoplasma pneumoniae, Bacillus subtilis, Staphylococcus aureus, Mycobacterium tuberculosis, Synechocystis sp., Aquifer aeolicus, Deinococcus radiodurans, Thermus thermophilus, and Thermus aquaticus; and corresponding residues of the largest subunits of human RNAP I, human RNAP II, and human RNAP III.

FIG. 1B illustrates sequence alignments for the bacterial RNAP homologous RNA-exit-channel amino-acid sequences within rpoC (.beta.') from Escherichia coli; and corresponding residues of Haemophilus influenzae, Vibrio cholerae, Pseudomonas aeruginosa, Treponema pallidum, Borrelia burgdorferi, fastidiosa, Camploacter jejuni, Neisseria meningitides, Rickettsia prowazekii, Chlamydia trachomatis, Mycoplasma pneumoniae, Bacillus subtilis, Staphylococcus aureus, Mycobacterium tuberculosis, Synechocystis sp., Aquifer aeolicus, Deinococcus radiodurans, Thermus thermophilus, and Thermus aquaticus; and corresponding residues of the largest subunits of human RNAP I, human RNAP II, and human RNAP III.

FIG. 2 illustrates a model of the three-dimensional structure of Thermus thermophilius RNAP showing the location of bacterial RNAP homologous RNA-exit-channel amino-acid sequence. The view is directly into the RNA-exit-channel of RNAP--toward the active-center cleft. Atomic coordinates are based on the crystallographic structure of Thermus thermophilius RNAP at 2.6 .ANG. resolution (Vassyleyev et al., 2002; PDB accession 1IW7; .sigma. subunit omitted for clarity).

FIG. 3 shows a polyacrylamide gel illustrating Lpm inhibition of formation of RNAP-promoter open complex.

Best mode of carrying out the invention

The present invention provides methods of designing specific inhibitors of bacterial RNAP, the enzyme responsible for transcription. The invention provides targets and methods for specific binding and inhibition of RNAP from bacterial species. The invention has applications in control of bacterial gene expression, control of bacterial growth, antibacterial chemistry, and antibacterial therapy.

Recently, crystallographic structures have been determined for bacterial RNAP and eukaryotic RNAP II (Zhang et al.,

Cell 98, 811-824; Cramer et al.,

Science 288, 640-649; Cramer et al.,

Science 292, 1863-1876; Ebright, R.

J. Mol. Biol. 304, 687-689; Gnatt et al.,

Science 292, 1876-1882; Murakami et al.,

Science 296, 1280-1284; Murakami et al.,

Science 296, 1285-1290; Vassylyev et al.,

Nature 417, 712-719; Bushnell et al.,

Science 303, 983-988; Westover et al.,

Science 303, 1014-1016). Based on these crystallographic structures, and biophysical and biochemical results, structural models have been proposed for transcription initiation and elongation complexes ((Naryshkin et al.,

Cell 101, 601-611; Kim et al.,

Science 288, 1418-1421; Korzheva et al.,

Science 289, 619-625; Ebright, R.

J. Mol. Biol. 304, 687-689; Gnatt et al.,

Science 292, 1876-1882; Mekler et al.,

Cell 108:599-614; Murakami et al.,

Science 296, 1280-1284; Murakami et al.,

Science 296, 1285-1290; Vassylyev et al.,

Nature 417, 712-719; Bushnell et al.,

Science 303, 983-988; Westover et al.,

Science 303, 1014-1016).).

The structural models include an approximately 30 .ANG. long, 15 .ANG. wide channel, known as the "RNA-exit-channel," that connects the RNAP active-center cleft to the RNAP exterior. In transcription initiation complexes, transcription initiation factors occupy this channel: i.e., initiation factor .sigma. region 3.2 (also known as the ".sigma.R3/.sigma.R4 linker" or ".sigma.3/.sigma.4 linker") in the case of bacterial transcription initiation complexes and transcription initiation factor IIB N-terminal domain in the case of eukaryotic RNAP II transcription initiation complexes. In transcription elongation complexes, the nascent RNA product occupies this channel.

The structural models imply that the RNAP RNA-exit-channel plays roles in biochemical activities important for function of RNAP in transcription initiation and elongation, including: interaction between RNAP and initiation factors, interaction between RNAP and DNA (which is modulated by initiation factors), interaction between RNAP and initiating NTPs (which is modulated by initiation factors), and interaction between RNAP and the nascent RNA product.

It has now been found, and is disclosed herein, that binding of a small molecule within the RNAP RNA-exit-channel inhibits at least two of these biochemical activities. Specifically, it has now been found, and is disclosed herein, that binding of a small molecule within the RNAP RNA-exit-channel disrupts RNAP-initiation-factor interactions and disrupts RNAP-DNA interactions.

The present invention includes the discovery that a region within the bacterial RNAP RNA-exit-channel comprising residues corresponding to, and alignable with, 1251, 1256, and 1321 of the .beta. subunit of RNAP from Escherichia coli and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli (the "homologous RNA-exit-channel amino-acid sequence" or "target"; FIG. 1) is a useful target for compounds that block transcription. The corresponding residues in RNAP from Bacillus subtilis RNAP are residues 1056, 1061, and 1126 of the .beta. subunit and residues 237-238 of the .beta.' subunit of RNAP (FIG. 1). It was found that these residues are invariant or nearly invariant in RNAP from bacterial species, but are radically different in RNAP from eukaryotic species (FIG. 1). It further was found that these residues form an approximately 10 .ANG. wide shallow pocket within the wall of the RNAP RNA-exit-channel (FIG. 2).

The target is located within the bacterial RNAP RNA-exit-channel, a .about.30 .ANG. long, .about.15 .ANG. wide tunnel that mediates multiple biochemical activities of bacterial RNAP, including: interaction with .sigma. region 3.2 in free RNAP holoenzyme, interaction with .sigma. region 3.2 in transcription initiation complexes, interaction with the nascent RNA product in transcription elongation complexes, and extrusion of the nascent RNA product in transcription elongation complexes. The location of the target within the bacterial RNAP RNA-exit-channel is such that binding to the target of a sufficiently large molecule would be predicted to block the RNA-exit-channel and thereby to inhibit some or all of the above-listed biochemical activities. The location of the target within the RNAP RNA-exit-channel also is such that binding to the target of a sufficiently large molecule would be predicted to interfere with interactions between RNAP and DNA (both indirectly, through disruption of RNAP-.sigma.-region-3.2 interactions, and directly through steric clash with the DNA template strand).

The target referred to above in Escherichia coli RNAP is similar in amino-acid sequence to that of most or all other species of bacterial RNAP, and is referred to herein as the "homologous bacterial RNAP RNA-exit-channel amino-acid sequence". (For example, amino acid residues 1251, 1256 and 1321 of the .beta. subunit and residues 248-249 and 337 of the .beta.' subunit of RNAP from Escherichia coli exhibit high similarity to residues 1056, 1061, and 1126 of the .beta. subunit and residues 237-238 and 326 of the .beta.' subunit of Bacillus subtilis RNAP (FIG. 1).) Thus, the discovery of a molecule that binds to the target and inhibits an activity associated with the RNA-exit-channel in Escherichia coli RNAP also is likely to bind to the target an inhibit an activity associated with the RNA-exit-channel in other species of bacterial RNAP. Therefore, molecules found to have antibiotic activity (through binding to the target and inhibiting an activity associated with the RNA-exit-channel) against Escherichia coli are likely to be found to have antibiotic activity against other bacterial species.

In contrast, the target differs radically in amino acid sequence between bacterial RNAP and eukaryotic RNAP, including human RNAP I, human RNAP II, and human RNAP III. This allows for the identification of molecules that bind, in a target-dependent fashion, to bacterial RNAP, but that do not bind, or that bind substantially less well, to eukaryotic RNAP. This also allows for the identification of molecules that inhibit, in a target-dependent fashion, an activity of to bacterial RNAP, but that do not inhibit, or that inhibit substantially less well, an activity of eukaryotic RNAP. This differentiation is important, because it permits the identification of bacterial-RNAP-selective binding molecules and bacteria-selective inhibitors.

The invention provides, by way of example only, a target region corresponding to, and alignable with, residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli; as well as corresponding residues of the .beta.' subunit of RNAP from Bacillus subtilis, Haemophilus influenzae, Vibrio cholerae, Pseudomonas aeruginosa, Treponema pallidum, Borrelia burgdorferi, Ayella fastidiosa, Campylobacter jejuni, Neisseria meningitidis, Rickettsia prowazekii, Thermotoga maritima, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, Synechocystis sp., Aquifex aeolicus, Deinococcus radiodurans, Thermus thermophilus, and Therms aquaticus. This target is the bacterial RNAP homologous RNA-exit-channel amino-acid sequence.

The invention also provides a molecule that binds to RNAP from a bacterial species, making specific interactions with at least one residue within the set of residues corresponding to, and alignable with, residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

The invention also provides a compound that inhibits enzymatic activity of RNAP from a bacterial species making specific interactions with at least one residue within the set of residues corresponding to, and alignable with, residues 1056, 1061, and 1126 of the .beta. subunit of RNAP and residues 237-238 of the 13' subunit of RNAP from Bacillus subtilis.

The invention provides identification of a inhibitory compound by screening of a chemical library for molecules that: (a) bind to RNAP from a bacterial species, and (b) do not bind, or bind less well, to a derivative of RNAP from a bacterial species that has at least one amino acid substitution, deletion, or insertion, in an homologous RNA-exit-channel amino-acid sequence.

The invention also provides identification of a inhibitory compound by screening of a chemical library for molecules that: (a) inhibit enzymatic activity of RNAP from a bacterial species, and (b) do not inhibit enzymatic activity, or inhibit enzymatic activity less well, of a derivative of RNAP from a bacterial species that has at least one amino acid substitution, deletion, or insertion, in a bacterial RNAP homologous RNA-exit-channel amino-acid sequence.

The invention also provides identification of an inhibitory compound by screening of a chemical library for molecules that compete with a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence containing a detectable group for binding to RNAP from a bacterial species.

The invention also provides identification of an inhibitory compound by screening of a chemical library for molecules that alter the interaction of a region 3.2 within RNAP from a bacterial species.

The invention also provides identification of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence by screening a chemical library for molecules that alter the interaction of RNA with RNAP from a bacterial species.

The invention also provides identification of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence by screening a chemical library for molecules that inhibit open-complex formation by RNAP from a bacterial species.

The invention also provides for use of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence to identify, isolate, and/or immobilize RNAP from a bacterial species.

The invention also provides for use of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence to control bacterial gene expression.

The invention also provides for use of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence to control bacterial growth.

The invention also provides for use of a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence as an antibacterial agent.

One preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that binds to RNAP from a bacterial species, but does not bind, or binds less well, to RNAP from a mammalian species.

Another preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that inhibits biochemical activity of RNAP from a bacterial species, but does not inhibit biochemical activity, or inhibits biochemical activity less well, of RNAP from a mammalian species.

Another preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that binds to and/or inhibits RNAP from a broad spectrum of bacterial species.

Another preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that binds to and/or inhibits RNAP from a broad spectrum of both Gram-negative bacterial species.

Another preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that binds to and/or inhibits RNAP from a broad spectrum of Gram-positive bacterial species.

Another preferred aspect of the invention provides for a molecule specific for a bacterial RNAP homologous RNA-exit-channel amino-acid sequence that binds to and/or inhibits RNAP from a narrow spectrum of bacterial species.

Another preferred aspect of the invention provides for a molecule that binds to and/or inhibits RNAP from Escherichia coli, making specific interactions with at least one residue within the set consisting of residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

Another preferred aspect of the invention provides for a molecule that binds to and/or inhibits RNAP from Bacillus subtilis, making specific interactions with at least one residue within the set consisting of residues 1056, 1061 and 1126 of the .beta. subunit and residues 237-238 and 326 of the .beta.' subunit of RNAP from Bacillus subtilis.

The present invention further relates to a method for identifying molecules that bind to the RNA-exit-channel with an assay for molecules that bind to RNAP in a RNA-exit-channel-specific fashion. In one embodiment, Escherichia coli RNAP, or a fragment thereof containing the RNA-exit-channel, is used as the test protein for binding, and a derivative of said RNAP or RNAP fragment having at least one of a substitution, an insertion, or a deletion within the RNA-exit-channel is used as the control protein for target-site specificity of binding. "Hits" may be analyzed for binding and inhibition of Gram-negative-bacterial RNAP, Gram-positive-bacterial RNAP, and eukaryotic RNAP I, RNAP III and RNAP III, in vivo and in vitro. "Hits" may also be characterized structurally by x-ray diffraction analysis of co-crystals with RNAP or an RNAP fragment containing the RNA-exit-channel.

The invention also provides strategies to identify small-molecule inhibitors from compound libraries. By way of example, two strategies are described as follows: (a) selection of molecules that bind to RNAP, or a fragment thereof, in a RNA-exit-channel-dependent fashion (affinity selection of phage-displayed linear and cyclic decapeptide libraries), and (b) screening for molecules that inhibit transcription in a RNA-exit-channel-dependent fashion (iterative deconvolution of solution-phase linear and cyclic D-hexapeptide libraries). In each case, the invention provides the use of a wild-type bacterial RNAP, or fragment thereof, as the test protein for binding/inhibition, and a derivative of bacterial RNAP, or a fragment thereof, having at least one of a substitution, an insertion, or a deletion within the RNA-exit-channel as the control protein for RNA-exit-channel-dependence of binding/inhibition.

The invention also provides for a method of identifying a compound for use as an inhibitor of bacterial RNAP comprising: analyzing a compound or a compound library, that involves docking to, modeling of, geometric calculations with, and/or energetic calculations with, a portion of the structure of an RNAP from a bacterial species comprising at least one residue within the set of residues corresponding to, and alignable with, the target.

The invention provides for at least four drug-discovery assay methods: a) screening based on binding of a compound within the RNA-exit-channel of a bacterial RNAP or fragment thereof; b) screening based on inhibition of an activity associated with the RNA-exit-channel of a bacterial RNAP or fragment thereof; c) screening based on displacement of a derivative of a region 3.2, containing a detectable group, from the RNA-exit-channel of a bacterial RNAP of fragment thereof; and d) screening based on displacement of a compound, containing a detectable group, from the RNA-exit-channel of a bacterial RNAP or a fragment thereof.

One of the embodiments of the present invention is an assay system designed to identify compounds that bind a bacterial RNAP, or a fragment thereof, in a manner that requires the RNA exit channel. The assay is designed to measure the binding of a compound to a determinant that includes at least one amino acid residue contained within a set of amino acid residues identifiable by sequence alignment and/or structure alignment as corresponding to amino-acid residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

One of the embodiments of the present invention is an assay system designed to identify compounds that inhibit an activity of a bacterial RNAP, a fragment thereof, in a manner that requires the RNA-exit-channel. The assay is designed to measure the inhibition of an activity, said inhibition involving the binding of a compound to a determinant that includes at least one amino acid residue contained within a set of amino acid residues identifiable by sequence alignment and/or structure alignment as corresponding to amino-acid residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

Another embodiment of the present invention is an assay designed to measure the binding of a compound to a bacterial RNAP derivative, or a fragment thereof, containing at least one amino acid substitution, insertion, or deletion within a set of amino acid residues identifiable by sequence alignment and/or structure alignment as corresponding to amino-acid residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

Another embodiment of the present invention is an assay designed to measure the inhibition of an activity of a bacterial RNAP derivative, or a fragment thereof, containing at least one amino acid substitution, insertion, or deletion within a set of amino acid residues identifiable by sequence alignment and/or structure alignment as corresponding to amino-acid residues 1251, 1256, and 1321 of the .beta. subunit and residues 248-249 of the .beta.' subunit of RNAP from Escherichia coli.

Isolation of RNAP:

The bacterial RNAP, or RNAP derivative, can be isolated from bacteria, produced by recombinant methods, or produced through in vitro protein synthesis. Various compounds can be introduced to determine whether a tested compound binds to, inhibits an activity of, or displaces a detectable-group containing molecule from, the bacterial RNAP or RNAP derivative in a RNA-exit-channel-dependent manner.

Tested compounds can include antibodies, peptides, and various chemical compounds. Additionally, with the known amino acid sequence for a particular RNAP, one of skill in the art could design specific inhibitors.

The assay can be performed in vivo or in vitro and thus does not necessarily require isolation of the RNAP.

The tested compounds can be chosen from chemical libraries, or a computer model can be used to choose compounds that are likely to be effective based on the known structure of the RNA-exit-channel and the structure of the compound.

The compounds can also be tested for competitive inhibition. Preferred strategies for identifying inhibitors include: 1) through affinity selection of phage-displayed linear and cyclic decapeptide libraries, and 2) through iterative deconvolution of solution-phase linear and cyclic D-hexapeptide libraries. One of wild-type Escherichia coli RNAP and wild-type Bacillus subtilis RNAP is the preferred test protein for binding and inhibition. One of a derivative of Escherichia coli RNAP having at least one substitution in the target and a derivative of Bacillus subtilis RNAP having at least one substitution in the target is the preferred control protein. Deconvolution essentially entails the resynthesis of that combinatorial pool or mixture that are found to be active in screening against a target of interest. Resynthesis may result in the generation of a set of smaller pools or mixtures, or a set of individual compounds. Rescreening and iterative deconvolution are performed until the individual compounds that are responsible for the activity observed in the screens of the parent mixtures are isolated.

Phage-Display Approach:

Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." (See

Science 249:386;

Science 249:404; and

Proc. Natl. Acad. Sci. 87:6378). The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface. The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's. Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates.

"Fusion phage" is filamentous bacteriophage vectors in which foreign antigenic determinants are cloned into phage gene III and displayed as part of the gene III protein (pill) at one tip of the virion. Fusion phage whose displayed determinant binds an antibody (Ab) can be selected from a vast background of nonbinding phage by affinity purification (AP) as follows: First, phage are reacted with biotinylated Ab (bio-Ab), then diluted and placed on a streptavidin-coated petri dish, thereby specifically attaching Ab-reactive phage to the plastic surface through the Ab-biotin-streptavidin bridge. Free phage is washed away, and bound phage eluted in acid and used to infect Escherichia coli cells. A single round of AP can enrich Ab-binding phage by as much as a factor of 10.sup.5 relative to unreactive phage; further enrichment is achieved by further rounds of AP after amplification on agar medium. Thus, Ab serves as a powerful selective agent favoring the target clones, so that vast numbers of phage can be surveyed.

The idea of using fusion phage to develop an "epitope library" (Parmley and G. P. Smith,

Gene 73:305) was inspired by the synthetic "mimotope" strategy of Geysen et al. (See Synthetic Peptides as Antigens; Ciba Foundation Symposium 119, R. Porter and J. Wheelan, Eds. (Wiley, New York. 1986), pp. 131-149). By synthesizing peptide mixtures on plastic pins and assessing their ability to bind an Ab against a protein antigen, these workers delineated a peptide that mimics a discontinuous epitope--an Ab-binding determinant composed of residues distant in the primary sequence but adjacent in the folded structure. They called these peptide mimics mimotopes. In this way, ligands can be discovered for an Ab whose specificity is not known in advance.

Fusion phage displaying short cloned peptides is infectious analogs of chemically synthesized mimotopes, with the key advantages of replicability and clonability. A large library of such phage--an "epitope library"--may display tens of millions of peptide epitopes. The peptides can in effect be individually surveyed for binding to an Ab or other binding protein by affinity purifying reactive phage from the library, propagating individual phage clones, and sequencing the relevant part of their DNA's to determine the amino acid sequences of their displayed peptides. A survey based on the epitope library undoubtedly would be imperfect because of bias introduced by the biology of the phage and other factors; still, it represents a powerful approach to the study of the specificity of Ab's and other binding proteins. (See Scott and Smith

Science 249:386; Devlin et al.,

Science 249:404; Ciwirla et al.,

Proc. Nat'l Acad. Sci. 87:6378; McLafferty et al.,

Gene 128:29; Alessandra et al.,

Gene 128:51; McConnell et al.,

Gene 151:115, which are incorporated herein by reference).

Iterative-Deconvolution and Positional-Scanning Approaches

See the following reference for a general discussion of iterative deconvolution: (Ostresh et al.,

Meths. Enzym. 267:220, which is incorporated herein by reference). The practical development of synthetic combinatorial libraries (SCLs) made up of tens of millions of compounds has proven to be a powerful source for the identification of novel biologically active compounds such as analgesics, antibacterials, antifungals, and enzyme inhibitors. (See Pinilla et al.,

Drug Dev. Res. 33:133; Pinilla et al.,

Pept. Sci. 37:221; Gallop et al.,

J. Med. Chem. 37:1233). In particular, a range of new compounds having potent antimicrobial and/or antifungal activities has been rapidly identified from pools of millions of compounds. (See Blondelle et al.,

J. Appl. Bacteriol. 78:39; Blondelle et al.,

Antimicrob. Agent Chemother. 38:2280; Ostresh et al.,

Proc. Nat'l. Acad. Sci. U.S.A. 91:11138; Houghten et al.,

Bio Techniques 13:412; Houghten et al.,

Nature 354:84).

Nonsupport-bound SCLs, originally composed of millions of peptides, are known to be usable in virtually any assay system (including those involving membrane-bound acceptors or whole cell organisms). In an expansion of SCL concepts and diversities, the original peptide SCLs have been transformed (i.e., peralkylated and/or exhaustively reduced) using a "libraries from libraries" approach (Ostresh et al.,

Proc. Natl. Acad. Sci. U.S.A. 91:11138; Dorner et al., in "Peptides 1994: Proceedings of the 23rd European Peptide Symposium" (H. L. S. Maia, ed.), p. 463. Escom, Leiden, 1995; and Cuervo et al., Id. at page 465) to yield peptidomimetic and organic libraries having entirely different physical, chemical, and biological properties relative to the peptide SCLs used as starting materials. The screening of such libraries has yielded active compounds derived from entirely different sequences than the active peptides previously identified from the starting SCLs using the same assay.

Two approaches can be employed for the structural deconvolution of active compounds from assay data using nonsupport-bound SCLs: the "iterative" approach and the "positional scanning" approach. In addition, two synthetic methods can be used for the incorporation of multiple functionalities at diverse positions within an SCL. As first illustrated for peptides, (See Houghten et al.,

Bio Techniques 13:412; and Houghten et al.,

Nature 354: 84, which are incorporated herein by reference). The first synthetic method, known as the "divide, couple, and recombine" (DCR) (Id.) or "split resin" (Lam et al.,

Nature 354:82) method, has typically been used with the iterative deconvolution approach. The second synthetic method, which involves the use of a predefined chemical ratio of protected amino acids at each coupling step for incorporation of mixture positions, Ostresh et al.,

Biopolymers 34:1681) has been developed for use with the positional scanning deconvolution process (Pinilla et al.,

BioTechniques 13:901). This latter method offers the advantage that both defined and mixture positions are easily incorporated at any position in a sequence.

These synthesis and deconvolution methods have been used to identify individual active compounds in a wide variety of SCLs and assays. (Pinilla et al.,

Drug Dev. Res. 33:133; Pinilla et al.,

Pept. Sci. 37:221). More specifically, individual compounds from nonsupport-bound SCLs have been identified which have potent antimicrobial activity against gram-positive bacteria (Staphylococcus aureus, Streptococcus sanguis), gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), and fungi (Candida albicans). The iterative deconvolution approach will be illustrated here for the preparation of a dual-defined position hexapeptide SCL, designated OOXXXX--NH.sub.2 (where O represents a defined amino acid, and X represents a mixture of amino acids) using the DCR method. The mixtures making up this library have been assayed for antimicrobial and/or antifungal activity (Blondelle et al.,

Trends Anal. Chem. 14:83; Houghten et al.,

Bio Techniques 13:412; and Houghten et al.,

Nature 354:84) in order to identify the first two amino acid residues of active hexapeptide sequences. The remaining four positions were then identified sequentially through an iterative process of synthesis and screening. This process can be completed in 6 to 10 weeks (four separate iterative synthesis steps are required). The positional scanning approach, involves the screening of separate single position SCLs to identify the most effective amino acids at each position of the sequence. When used in concert, this information can be used to identify individual active sequences. This process can be completed in approximately 2 weeks (only one synthesis step is required for confirmation of activity).

Both iterative and positional scanning peptide SCLs are used as starting materials for the generation of peptidomimetic SCLs using the "libraries from libraries" approach.

The description continues in the full USPTO document.